Collagen fibril disruption occurs early in primary guinea pig knee osteoarthritis  J.L. Huebner, J.M. Williams, M. Deberg, Y. Henrotin, V.B. Kraus  Osteoarthritis and Cartilage  Volume 18, Issue 3, Pages 397-405 (March 2010) DOI: 10.1016/j.joca.2009.09.011 Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Schematic representation of the specific type II collagen epitopes measured. The epitopes measured in sera result from the three types of collagen degradation: collagenase cleavage (C2C), denaturation (Coll2-1), and MMP-generated collagen telopeptides (CTX-II). Osteoarthritis and Cartilage 2010 18, 397-405DOI: (10.1016/j.joca.2009.09.011) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Histological damage of the guinea pig knee joints by compartment. (A) Mean cartilage structure scores and (B) mean proteoglycan loss in each of the compartments (LF; LT; MF and MT) of the guinea pig knee from 2 to 18 months of age. Differences in histological scores over time at various sites within the joint were calculated by the one-way ANOVA non-parametric Kruskal–Wallis test. P values<0.05 are considered significant. Osteoarthritis and Cartilage 2010 18, 397-405DOI: (10.1016/j.joca.2009.09.011) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Representative coronal sections of whole joints depicting variations in percent areas of normal birefringence in the tibial compartment. Appearance of the tibial surface at 3 weeks (A) showing a typical pattern of birefringence; normal birefringence on the surface (zone 1; red arrowheads), no birefringence in zone 2 (red asterisks) and normal birefringence in zone 3; at 2 months of age (B) there is normal birefringence on the surface (zone 1; red arrowheads), no birefringence in zone 2 (red asterisks) with some reduction of normal birefringence in zone 3; at 4 months of age (C) there is a loss of normal birefringence on the surface (zone 1), no birefringence in zone 2 and a mixture of normal birefringence (dashed circle) and loss of normal birefringence is noted in zone 3; at 7 months of age (D) there is loss of normal birefringence on the surface (zone 1), surface disruption (red asterisk), no birefringence in zone 2, and loss of normal birefringence is noted in zone 3; at 10 months of age (E) there is loss of normal birefringence on the surface (zone 1) and evidence of surface disruptions (red asterisks), no birefringence in zone 2, and a mixture of normal birefringence (dashed circle) and loss of birefringence is noted in zone 3; at 12 months of age (F) there is a complete loss of normal birefringence on the surface (zone 1) followed by birefringence in zone 2 (dashed circle) and again a mixture of normal (white dashed circle) and a loss of normal birefringence in zone 3. The solid white line approximates the cartilage bone interface. Original magnification 40x. Osteoarthritis and Cartilage 2010 18, 397-405DOI: (10.1016/j.joca.2009.09.011) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Disruption of the collagen network integrity with age. Collagen birefringence was scored semi-quantitatively from picrosirius red stained sections of Hartley knee joints, aged 2 months to 18 months. The % loss of normal birefringence was estimated based upon comparison with normal birefringence as seen in a reference area. Percentage loss of birefringence for each of the four compartments is shown (LF: ; LT: ; MF: and MT: ). Osteoarthritis and Cartilage 2010 18, 397-405DOI: (10.1016/j.joca.2009.09.011) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Immunohistochemical expression of collagenase-generated collagen degradation. Immunostaining of representative guinea pig knee sections with monoclonal antibody (mAb) 9A4. The MT compartment (A) of a 2-month-old guinea pig depicts cleavage by collagenase in the superficial zone of the compartment which preceded histological changes associated with OA. In contrast, there is more uniform extracellular staining throughout the articular cartilage of the LT compartment at 2 months of age (B). MT compartment of 4 month old (C) and 7 month old (E) stained with monoclonal antibody 9A4 show more intense staining of the superficial zone and areas of cartilage degradation, respectively. Arrows indicate the pericellular staining of chondrocytes throughout the middle and deep zones of the articular cartilage. MT compartment of a 2-month-old guinea pig knee stained with secondary antibody only (negative control) (D). Extracellular staining of the epiphyseal plate was evident (F). Magnification bars: (A–E; 40×=50μM and F; 10×=100μM). Osteoarthritis and Cartilage 2010 18, 397-405DOI: (10.1016/j.joca.2009.09.011) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Immunohistochemical expression of Coll2-1 in guinea pig knee sections. The MT (A) and LT (B) compartments of a 2-month-old guinea pig demonstrates the presence of the Coll2-1 epitope intracellularly in chondrocytes of the superficial, middle, and deep zones, but no extracellular staining of the cartilage matrix. MT compartment of a 4 month old (C) guinea pig shows no staining in the superficial zone devoid of chondrocytes, however in contrast, arrows indicate the staining of the fibrillated cartilage in the 7-month-old section (E). Arrows indicate the intracellular staining of chondrocytes throughout the deep zone of the articular cartilage. MT compartment of a 2-month-old guinea pig knee stained with secondary antibody only (negative control) (D). No extracellular staining of the epiphyseal plate was evident (F). Magnification bars: (A–E; 40×=50μM and F; 10×=100μM). Osteoarthritis and Cartilage 2010 18, 397-405DOI: (10.1016/j.joca.2009.09.011) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 Comparison of type II collagen degradation biomarkers. Measurement of type II collagen degradation biomarkers C2C, Coll2-1, and CTX-II in guinea pig sera from animals aged 3 weeks to 18 months. Results were expressed as mean±SD. Osteoarthritis and Cartilage 2010 18, 397-405DOI: (10.1016/j.joca.2009.09.011) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 8 Distribution of serum biomarker levels by quartiles of total histological score. Total histological scores were divided into quartiles (Q1: 0–0; Q2: 0–9; Q3: 10–18; Q4: 19–32) to examine the distribution of levels of serum biomarker by severity of histological OA. *P<0.05, **P<0.01, ***P<0.001. Osteoarthritis and Cartilage 2010 18, 397-405DOI: (10.1016/j.joca.2009.09.011) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions